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Session A2: Immortalization Versus Redifferentiation of Cell LinesChairs: Jose Castell (Spain) and William Smith (USA) A2: The Use of Cultured Human Keratinocytes in Dermatological Investigations Chemical injury to the skin can span a wide range of pathological parameters and cell types. While barrier function usually resides in the upper strata of the epidermis, many of the toxic manifestations in skin center around the basal cells and their interactions with other cells in both the epidermis and dermis. The human epidermal keratinocyte (HEK) in culture is the in vitro equivalent of the basal epidermal cell. HEK are often used for analysis of human dermatopathological processes as an alternative to whole animal studies. HEK are available either as primary isolates from human skin biopsies or from commercial sources as primary or immortalized cultures. The selection of an appropriate culture model is not only dependent on the end-point of the assay, but can be subject to considerations of cost and assay reproducibility. For the past few years, our laboratory has studied the toxicity of the chemical blistering agent sulfur mustard in HEK. This report will discuss parameters that must be considered when using HEK in studies of cell proliferation, DNA damage and repair, activation of inflammatory processes, toxicogenomics, and other cytotoxic phenomenon. A2: Can Hepatoma Cell Lines be Redifferentiated to be Useful in Drug Metabolism Studies? Knowledge of metabolism, enzymes so far involved, and potential enzyme-inhibiting or enzyme-inducing properties of new compounds is a key issue in drug development. Primary cultured hepatocytes, CYP engineered cells, and hepatoma cell lines are presently being used for this purpose, but only primary cultures can produce a metabolic profile of a drug similar to that found in vivo and respond to inducers. Because of their limited access, alternatives to replace human hepatocytes are currently being explored, including immortalization of hepatocytes using different strategies (i.e. SV40 T-large antigen, conditionally immortalized hepatocytes, transfection with c-myc, cH- ras, N-ras oncogenes, transgenic animals overexpressing growth factors or oncogenes and cre-lox recombination/excision, etc.). However, none of the resulting cells has the desirable phenotypic characteristics to replace primary cultures in drug metabolism studies. We investigated why these differentiated human hepatomas do not express CYP genes and found that the levels of certain key transcription factors clearly differ from those found in hepatocytes. It was then conceivable that re-expression of one (or more) of these transcription factors could lead to an efficient transcription of CYP genes. The feasibility of this hypothesis was demonstrated by co-transfecting HepG2 cells with C/EBP-sigma, HNF-1, HNF-3, and HNF-4 isoforms and examining the expression of the most relevant human CYP's in transfected cells. A2: Transfection of Hybridoma Cell Lines with an Anti-Apoptosis Gene Increases Viability and In Vitro Monoclonal Antibody Production In order to improve in vitro production of monoclonal antibodies (MAb), we transfected a hybridoma cell line (552) with the cDNA encoding an anti-apoptosis gene, BCl-2 delta. After transfection, cells were distributed at low concentrations to 96 well plates in selection media (G418). Individual clones were selected after 10 days of growth and expanded. Western blot analysis revealed high levels of BCl-2 delta expression in 2 of the 6 clones selected. Viability analysis of non-transfected, vector alone and vector-BCl-2 delta transfected 552 cells under conditions typical for batch MAb production (high density and acidity) revealed that the BCl-2 delta-expressing clone retained a higher level of viability for a longer period of time and produced approximately twice the amount of mouse MAbs when compared to the controls. These results indicate that transfection of hybridoma cell lines with BCl-2 delta may result in increased MAb yields and may make in vitro production methods more attractive. Work continuing in our laboratory with other hybridoma cell lines will be presented and the use of transgenic mice to improve in vitro MAb production yields will be discussed. A2: Cell Signalling in Trichostatin A-Treated Primary Hepatocyte Cultures: Effect on Proliferation and Apoptosis HDAC inhibitors, with Trichostatin A (TSA) as a prototype, are currently under extensive investigation for the treatment of hyperproliferative diseases. Many studies with respect to the molecular events associated with TSA-induced growth arrest and apoptosis have already been performed in neoplasm-derived transformed cells. Hepatocytes, however, are of major interest for toxicological and metabolism-linked studies, because they are the major sites of xenobiotic biotransformation. Therefore, we investigated several cell cycle factors and apoptosis-regulating proteins in primary cultured rat hepatocytes exposed to TSA. It was found that TSA-induced growth arrest in primary hepatocytes was accompanied by increased p21waf1/cip1 expression along with accumulation of hyperacetylated histones H3 and H4. The p21waf1/cip1 induction in rat hepatocytes is accompanied by downregulation of CDK1 protein expression without affecting the CDK2 protein level. In addition, in the presence of TSA, CDK1-, and 2-dependent histone H1 kinase activities, characterizing the G1/S boundary, strongly decrease at 72 hours. Furthermore, in the presence of TSA, the expression of c-myc, a marker for the early G1 phase, is markedly delayed and followed by a complete disappearance of its expression. This is in contrast with untreated primary hepatocytes where c-myc is stably expressed during the whole culture period. Concomitantly, caspase-8 and the pro-apoptotic protein Bax were downregulated in TSA-treated hepatocytes. The effect of TSA on Bcl-2 expression as well as the p53 response and E2F transcription are currently being investigated to be able to connect c-myc with the apoptosis pathway. Altogether, these results clearly show that HDAC regulates gene transcription at specific promoters of cell cycle and apoptosis genes in primary cultures of rat hepatocytes. A2: Use of Genetically Engineered PC12 Cell Lines for Detecting Subchronic p53-Mediated Cell Death Previous experimental work performed at ECVAM resulted in the development of a genetically-modified PC12 cell line, which displays different sensitivities toward apoptotic cell death. Expression of the pro-apoptpotic gene, wt p53, is controlled by the use of defined media. The present study was undertaken to evaluate their applicability as a test system for studying p53-mediated cell death in subchronic neurotoxicity testing. In a first step, cells were treated with nerve growth factor (NGF) to induce cell differentiation and the acquisition of neuron-like characteristics. The expression of the p53 protein was found to increase with increasing concentrations of NGF on the different days tested (1 to 10 days). NGF treatment also resulted in the development of neuritis. However, the doubling time of the cells remained unchanged, as compared to that of untreated cells. Cells were further subchronically treated for 10 days with methylmercury, a known neurotoxicant, and the type of cell death occurring was characterized. It was found that the genetically modified PC12 cell lines could display both apoptotic and necrotic mechanisms of cell death.
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